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Resveratrol Improves Intrahepatic Endothelial Dysfunction and Reduces Hepatic Fibrosis and Portal Pressure in Cirrhotic Rats
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Journal of Hepatology 4 December 2012
Article in Press
Marco Di Pascoli, Marta Div., Aina Rodr.guez-Vilarrupla, Eugenio Rosado,
Jorge Gracia-Sancho, Marina Vilaseca, Jaume Bosch, Joan Carles Garc.a-
Pagn
"Resveratrol administration reduces portal pressure, hepatic stellate cell activation and liver fibrosis, and improves hepatic endothelial dysfunction in cirrhotic rats, suggesting it may be a useful dietary supplement in the treatment of portal hypertension in patients with cirrhosis.......In conclusion, our data show that chronic resveratrol administration to cirrhotic rats reduces portal pressure both by causing a regression of liver fibrosis and by correcting hepatic endothelial dysfunction, without affecting systemic hemodynamics. Due to these properties and its low toxicity, resveratrol, which is widely available, may be a useful supplement in the treatment of patients with cirrhosis and portal hypertension."
"we could speculate that in our setting the antifibrotic effect of resveratrol may be due, at least partly, to its anti-oxidant activity [35]. Indeed, it has been shown that resveratrol inhibits the activation of NF-kappaB, which promotes the transcription of several cytokines including the pro-fibrogenic TGF-ß [18, 36, 37]. In that regard, we have also demonstrated that resveratrol treatment produces a significant reduction in the NFB and TGF gene expression, suggesting that resveratrol is able to reduce fibrosis by reducing profibrogenic stimuli in cirrhotic rat livers"
"Resveratrol (3,5,4'-trihydroxystilbene) is a natural polyphenolic flavonoid found in a large amount of plant species, including grapes and their derivatives, berries and nuts. It has been suggested to have important health benefits attributed to its demonstrated anti-oxidant, anti-neoplastic, anti-inflammatory and anti-platelet aggregation activities [8-11]. Specifically, in different experimental models resveratrol improves vascular dysfunction"
Abstract
Background and Aims
Resveratrol, a polyphenol found in a variety of fruits, exerts a wide range of beneficial effects on the endothelium, regulates multiple vasoactive substances and decreases oxidative stress, factors involved in the pathophysiology of portal hypertension. Our study aimed at evaluating the effects of resveratrol on hepatic and systemic hemodynamics, hepatic endothelial dysfunction and hepatic fibrosis in CCl4-cirrhotic rats.
Methods
Resveratrol (10 and 20 mg/kg/day) or its vehicle were administered to cirrhotic rats for two weeks and hepatic and systemic hemodynamics were measured. Moreover, we evaluated endothelial function by dose-relaxation curves to acetylcholine, hepatic NO bioavailability and TXA2 production. We also evaluated liver fibrosis by Sirius Red staining of liver sections, collagen-1, NFkB, TGFß mRNA expression, and desmin and α-smooth muscle actin (α-SMA) protein expression, as a surrogate of hepatic stellate cell activation.
Results
Resveratrol administration significantly decreased portal pressure compared to vehicle (12.1±0.9 vs 14.3±2.2 mmHg; p<0.05) without significant changes in systemic hemodynamics. Reduction in portal pressure was associated with an improved vasodilatory response to acetylcholine, with decreased TXA2 production, increased endothelial NO and with a significant reduction in liver fibrosis. The decrease in hepatic fibrosis was associated with a reduced collagen-1, TGFß, NFκB mRNA expression and desmin and α-SMA protein expression.
Conclusions
Resveratrol administration reduces portal pressure, hepatic stellate cell activation and liver fibrosis, and improves hepatic endothelial dysfunction in cirrhotic rats, suggesting it may be a useful dietary supplement in the treatment of portal hypertension in patients with cirrhosis.
Introduction:
In cirrhosis, the initial factor determining the onset of portal hypertension is the increase in intrahepatic vascular resistance. This is not only due to
morphological changes resulting from the chronic liver inflammation and fibrosis, but also to reversible functional alterations, including an exaggerated
response of the porto-hepatic vascular bed to vasoconstrictors and a deficient response to vasodilators [1]. A decreased nitric oxide (NO) availability and an
increase in cyclooxygenase-1 (COX-1)-derived prostanoids within the liver play a major role in the pathogenesis of these dynamic alterations [2-5].
Reduced NO availability has been shown to be in part due to an increase scavenging by superoxide (O2-) and different strategies aimed to reduce O2-
levels [6, 7] such as superoxide dismutase (SOD) gene transfer are able to reduce portal pressure in experimental models of cirrhosis in the rat.
Resveratrol (3,5,4'-trihydroxystilbene) is a natural polyphenolic flavonoid found in a large amount of plant species, including grapes and their derivatives, berries and nuts. It has been suggested to have important health benefits attributed to its demonstrated anti-oxidant, anti-neoplastic, anti-inflammatory and anti-platelet aggregation activities [8-11]. Specifically, in different experimental models resveratrol improves vascular dysfunction, an effect that is attributed to its ability to reduce oxidative stress, to upregulate endothelial nitric oxide synthase (eNOS) expression and activity, and to inhibit COX-1 activity [12-15].
Resveratrol has been shown to exert anti-oxidant effects in experimental models of liver injury induced by ischemia/reperfusion and ethanol by inducing the enzymatic activity of SOD and catalase [16, 17], and to attenuate fibrosis development when co-administrated with CCl4 to rats [18]. Additionally, resveratrol reduces the hepatotoxicity induced by acetaminophen, ethanol and carbone tetrachloride (CCl4), and prevents liver damage due to ischemiareperfusion, irradiation and high fat diet [19]. Overall, we hypothesized that resveratrol may exert beneficial effects in the pathophysiological mechanisms involved in the development of portal hypertension in cirrhosis
Therefore, the aim of the present study was to investigate the effects of chronic administration of resveratrol in CCl4-cirrhotic rats with portal hypertension.
Discussion:
In cirrhosis, an increase in hepatic vascular resistance to portal blood flow is the primary factor in the development of portal hypertension [3]. Therefore, it is of great interest to develop therapeutic strategies aimed at decreasing portal pressure by reducing hepatic vascular resistance. Resveratrol is a natural substance with many biologic functions; among others, it induces antioxidant enzymes, increases NO bioavailability and inhibits the production of inflammatory factors [33].
The main finding of the present study is that in established cirrhotic rats with severe portal hypertension, the oral administration of resveratrol for two weeks reduces portal pressure, without affecting portal blood flow, indicating an effective reduction in hepatic vascular resistance. This beneficial effect on portal pressure was confirmed when a double dose of resveratrol was used. Remarkably, the beneficial effect of resveratrol on portal pressure occurred in the absence of deleterious effects on systemic hemodynamics, as shown by the absence of significant changes in mean arterial pressure, heart rate and superior mesenteric artery blood flow.
Our results further suggest that the reduction in hepatic resistance induced by resveratrol was the result of both amelioration of the liver architectural abnormalities and an improvement in endothelial dysfunction. Indeed, resveratrol significantly reduced liver fibrosis, as shown by the reduction in fibrosis area on Sirius Red stained liver sections and the decrease in collagen I mRNA expression, an effect that was not observed in cirrhotic rats treated with vehicle, demonstrating that this was due to an enhancement by resveratrol of the regression of fibrosis that occurs after ceasing CCl4 administration. Our results showing a decline in both desmin and SMA expression in liver tissue suggests that a decrease in HSC activation may be mostly due to the consequence of apoptosis of activated HSC. This suggestion is further supported by our findings in LX2 cells showing that resveratrol significantly decreases collagen I and SMA gene expression in association with an increase in Bad protein expression, a marker of apoptosis induction.
It has been well documented that a reduction of oxidative stress can deactivate mechanisms leading to liver fibrosis [34]. Therefore, we could speculate that in our setting the antifibrotic effect of resveratrol may be due, at least partly, to its anti-oxidant activity [35]. Indeed, it has been shown that resveratrol inhibits the activation of NF-kappaB, which promotes the transcription of several cytokines including the pro-fibrogenic TGF-ß [18, 36, 37]. In that regard, we have also demonstrated that resveratrol treatment produces a significant reduction in the NFB and TGF gene expression, suggesting that resveratrol is able to reduce fibrosis by reducing profibrogenic stimuli in cirrhotic rat livers. Whatever the mechanism, these effects on liver fibrosis were not boosted by higher doses of resveratrol, since similar reductions in fibrosis were observed with 10 and 20 mg/kg bw/day.
Cirrhotic livers of rats treated with resveratrol had a reduction in O2- levels. Resveratrol itself, as a polyphenolic compound, has been shown to scavenge hydroxyl, O2- and other radicals [38, 39]. Moreover, resveratrol have also indirect antioxidant effects by upregulating different endogenous cellular antioxidant systems, such as SOD, catalase, glutathione peroxidase,....and by inhibition of enzymatic systems involved in ROS formation, like NAPDH oxidase [40, 41]. Although decreased scavenging of O2- by diminished SOD activity has been reported as one of the causes of the increased oxidative stress in cirrhotic livers [24], we did not find any effect of resveratrol treatment on SOD activity. Therefore, we think that the observed is a direct antioxidant effect of resveratrol although we can not discard that other mechanisms different from SOD activity increase could be implicated.
Resveratrol administration also improved endothelial dysfunction. We have previously demonstrated that reduced NO bioavailability and a COX-1-dependent increase in TXA2 are the main factors mediating the endothelial dysfunction of cirrhotic rat livers [5, 42, 43]. Resveratrol has been shown to act as a peroxidase-mediated inactivator of COX-1 [15] and, in agreement with that, we found that resveratrol markedly inhibited TXA2 production in whole tissue. We also observed that resveratrol could attenuate the increase in TXA2 levels induced by AA administration in SEC-CH, confirming the role of COX-1-derived prostanoids in the improvement of endothelial dysfunction by resveratrol. In addition, resveratrol treatment promoted a significant increase in NO bioavailability in endothelial cells isolated from cirrhotic livers. Taken together, these results suggest that resveratrol may improve endothelial dysfunction by increasing endothelial NO and reducing endothelial TXA2. Besides, it is highly likely that the improvement in liver fibrosis produced by resveratrol itself may play a major role in the recovery of endothelial function. Furthermore, we herein show that resveratrol reduces the number of CD68- positive cells (34% decrease) in cirrhotic livers. This finding is in agreement with a published study showing that resveratrol may attenuate inflammatory infiltration [37], an effect that may represent an additional benefit of resveratrol treatment.
In conclusion, our data show that chronic resveratrol administration to cirrhotic rats reduces portal pressure both by causing a regression of liver fibrosis and by correcting hepatic endothelial dysfunction, without affecting systemic hemodynamics. Due to these properties and its low toxicity, resveratrol, which is widely available, may be a useful supplement in the treatment of patients with cirrhosis and portal hypertension.
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